In 1975 Kohler and Milstein demonstrated that individual clones of normal antibody-secreting cells could be fused with myeloma cells to produce a continuous cell line or culture which secreted monoclonal antibody to sheep red blood cells. Nature. Vol. 256,495 (1975). Since then numerous publications and patents have appeared describing the production of monoclonal antibodies to a variety of antigens and haptens. See, for example, "Monoclonal Antibodies: A Powerful New Tool in Biology and Medicine," Yelton, D. E. and Schaff, M. D., Ann. Rev. Biochem. 50:657-80, 1981; "Antibody Production by Hybridomas," Goding, James W., Journal of Immunological Methods. 39:285-308 (1980).
In monoclonal antibody production, each cell synthesizes a homogeneous or monoclonal immunoglobulin that represents one of the many antibodies produced by the immunized animal. For example, U.S. Pat. No. 4,521,540 discloses the production of hybridoma cell lines which produce monoclonal antibodies to theophylline. PCT Application No. GB85/00465 disclosed monoclonal antibodies to the genus Treponema. In particular, the monoclonal antibodies disclosed in this reference are specific to Treponema pallidum I, II and III; Treponema phagedenis; Treponema macrodentium or T. dentium: Treponema pertenue; Treponema cuniculi; Treponema refrigens; Treponema macrodenin: and Treponema mucosum. Additionally this reference discloses a monoclonal antibody cross-reactive with an antigen for each species of the genus Treponema.
Presently, diagnostic test kits using monoclonal antibodies have proved to be very useful due to their specificity and ability to produce reasonably accurate results. No such test kit is presently available, however, which accurately identifies the presence of oral disease, i.e., periodontitis, gingivitis and other diseases which evidence inflammation and subsequent loss of bone and gum tissue in the mouth. One reason for this is the difficulty in determining which organisms are the best indicators or causes of oral disease. Efforts in the development of a semi-quantitative diagnostic test useful for the detection of periodontal disease have focused on forming reagents to detect certain species of Treponema. A number of different bacterial organisms have been implicated in the disease process, but recently certain species of Treponema, a type of spirochete organism, have been shown to increase significantly in total microbial population as the disease progresses. (See Journal of Periodontology 53:550-556; 36:177-187; 38:379-386; 46:10-26; and Journal of Clinical Periodontology 8:122-138.) This organism is believed to play a major role in the oral disease process.
The human mouth provides a media rich in protein and amino acids, as well as other organic acids and inorganic substrates necessary for the growth and support of organisms. This complex environment necessitates development of reagents which are specific for and react only to the organisms of interest. Antibodies have the ability to recognize and bind specifically to the organisms used to generate them. For this reason, Treponema reactive antibodies were chosen as the reagents to be used in the development of the inventive assay and diagnostic tests which are useful in diagnostic test kits. This invention describes the formation of a polyclonal murine hybridoma cell line, subsequent cloning to obtain a monoclonal antibody secreting cell line and the reactivities of these novel antibodies. A method of using these monoclonal antibodies to diagnose the presence of the Treponema species described.